Biotechnology Reagents (IGFs) >> Product Support >> Application Notes >> #4030: Fish IGF-I antiserum for detection of IGF-I by RIA in a variety of finfish species

The antiserum (Product Code: PAF1) was obtained from rabbits immunized with recombinant barramundi (Lates calcarifer) IGF-I conjugated to ovalbumin. Supplied in lyophilized form, each vial contains approximately 10 µl of the antiserum. The antiserum is stable for at least 2 years at 2-4°C in a lyophilized state.

The antiserum is equipotent with [¹²⁵I]-labelled barramundi, bream (Pagrus auratus), salmon/trout (Oncorhynchus & Salmo sp.), tilapia (Oreochromis mossambicus) and tuna (Thunnus maccoyii) IGF-I tracer (80 Ci/g).

< 1% cross-reactivity with human IGF-I and human IGF-II.
No cross-reactivity with salmon insulin.

1. Recommended handling for use in Radioimmunoassay (RIA):

For radioimmunoassay we recommend the antiserum be used at a final dilution of 1/30,000. At this dilution the antiserum binds 20-25% of [¹²⁵I] Fish IGF-I tracer (80 Ci/g). Half-maximal inhibition (ED₅₀) of radioligand binding is produced by 2.5 ng/ml and the limit of detection is ~ 0.25 ng/ml. Each vial contains sufficient antiserum for approximately 1,000 RIA assay tubes.

Procedure:

The vial contents are dissolved by the addition of 500 µl of RIA buffer (30 mM sodium phosphate pH 7.5 containing 10mM EDTA, 0.2 g/l protamine sulphate, 0.5 ml Tween-20 and 0.25 g/l sodium azide) to the vial to give a 1/50 serum dilution.
After reconstitution, prepare a working solution by diluting the antiserum a further 1:50 with RIA buffer to give a 1/2,500 working solution. Working solutions are stable for 5 days at 4°C. The remaining 1/50 dilution should be aliquoted and stored at -20°C.

25 µl of the 1/2,500 working solution is added to each RIA tube (except the antibody-negative "blanks") in a final incubation volume of 0.3 ml giving a final dilution of antiserum of 1/30,000. Each RIA tube also contains approximately 20,000 cpm of [¹²⁵I] Fish IGF-I (80 Ci/g) (no more than 3 weeks old). Bound radioactivity is precipitated using conventional centrifugation procedures after addition of anti-rabbit antiserum, rabbit IgG and chilled PEG.

See Protocols:
#3001 Procedure for the Iodination of IGFs.

#3002 Determination of IGF-I or IGF-II in a Range of Species by Radioimmunoassay (RIA).

NOTE: When measuring IGF levels in Fish do NOT use the Novozymes GroPep Goat anti-Rabbit gamma globulin (IgG) (Catalog Code PSA1). The secondary antibody we recommend when measuring Fish IGFs is:

Chemicon sheep anti-rabbit IgG (IgG Fraction) (Catalog No AB7130)
Use 50 ul of a 1:20 dilution in RIA buffer per assay tube.

The contact details for Chemicon are:

uk@chemicon.com

www.chemicon.com


Results:

The relative potencies of the following peptides at inhibiting the binding of [¹²⁵I]salmon / trout IGF-I to this antiserum were found to be:

recombinant human IGF-I < 0.5%
recombinant human IGF-II < 1%
salmon insulin < 0.01%

* NOT FOR USE IN HUMANS


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